Procedure of the Immunohistochemistry and Laser Scanning

Immunohistochemical procedures were performed essentially as described by JURKEVICH et al. (1999). Briefly, sections were washed six times in 0.02 M PBS at room temperature. To reduce the nonspecific binding sites sections were incubated for 30 min in 0.02 M PBS containing 10% normal goat serum (Dako, Hamburg) and 0.2 % Triton X-100. Thereafter the sections were incubated at 4°C with rabbit polyclonal antibody against Arg-vasotocin diluted 1:30000 and guinea pig antibody against galanin (Peninsula laboratories, California) diluted 1:600 in 0.02 M PBS with 1% normal goat serum, 0.2% Triton X-100 and 0.1% sodium azide for 22 hours at 4°C. The sections were then rinsed in 0,02 M PBS and incubated with goat anti-rabbit IgG coupled with ALEXA 555 (Vector laboratories, Burlingame, TI-1000) diluted 1:400 and goat anti-guinea pig IgG coupled with fluorescein (FITC, Vector, FI-7000) diluted 1:400 in 0,02 M PBS with 0.2% Triton X-100, 1% normal goat serum and 0.1% sodium azide in the dark at room temperature for 90 min. After washing, the sections were rinsed again in 0.02 M PBS with 0.2% Triton X-100 and mounted on gelatine coated slides with vectashield mounting medium (Vector, H-1000) and then sealed with coverslips using colorless nail polish. The slides were stored at 4°C and protected from the light until confocal imaging microscopy was performed as multitracking of FITC (galanin) and ALEXA 555 (AVT). Argon laser (488 nm) exited for FITC and emission was seleted with bandpass of 505 to 520 nm. Helium/ Neon green laser (543nm) exited for Alexa 555 and emission was selected

with long pass of 585 nm. Optical slices (of 3 to 4 consecutive 40 µm sections from each brain) were focused in Z direction to 2 µm using a 20x objective with 512 pixels and to 1 µm in Z direction using a 40x objective. Number of cells expressing one or both of peptides were counted on 3D projection of 1 µm optical slices in total stacks of 30 µm thick central sections of 325 square µm.

The obtained digital data were transferred to the photoshop 6.0 software (Adobe San Jose, CA) for the preparation of the images for the presentation.

The AVT antibody was kindly provided by Dr. D. Gray (Max Planck Institute for Physiological and Clinical Research, Bad Nauheim, Germany). This antiserum and galanin antiserum used in this experiment have been successfully used in other chicken brain immunohistochemical studies conducted at the same time or previously in the Department of Functional Genomics and Bioregulation (BARTH et al. 1997;

JURKEVICH et al. 1997; KLEIN et al. 2006). All necessary control incubations such as preincubation of galanin antiserum (produced against full-length galanin) with

porcine galanin, change of the order of the incubation with primary antibodies (KLEIN et al. 2006) were conducted. The primary antibodies were omitted in some of

the incubations to control the background. The galanin antibody used in this experiment has been shown to have no crossreactivity to vasoactive intestinal peptide, secretin and human insulin.

The confocal laser scanning microscopy is routinely performed in the Department of Functional Genomics and Bioregulation. The instrument used in this study is LSM 510 (Carl Zeiss, Göttingen) and scanning is performed by servo-controlled galvanometers and the scanning time is 2 sec/ field of 512X512 pixels and a microprocessor controls the scanning process. After digitization, the signal is led to a frame memory the read-out of which is in video frequency. Consequently the image on the monitor is stationary provided (HELL 1996). A continuously variable magnification can be obtained by a zoom unit, the laser scan microscope was used with a conventional as well as with laser light source (HELL and STELZER 1992).

Both incident illumination (for reflectance and fluorescence) and transmitted illumination for absorbance, phase- and differential interference contrast) was obtained with laser source. To provide better focusing and searching possibilities in

Materials and Methods

33

the preparation and to allow investigation of double-stained probes, conventional epi-illumination device has been added to the laser scanner. An example (HELL 1997) of the separation of blue (460 nm) from green (530 nm) light is given in figure 5.

Fig. 5: Excitation and emission spectra of fluorescein isothiocyanate (FITC).

Wavelength of excitation; solid line and emission; dotted line (modified from SCHRADER and HELL 1996).

Similar chromatic beam splitters (CBS) as a dichroic mirrors exist for separation of other regions of the light spectrum, from the UV (300nm) to the red (700nm). Figure 6 demonstrates an example of an absorption and an emission spectrum. At a given wavelength, the most intense fluorescence can be obtained when the specimen is irradiated with a wavelength close to the peak of the excitation of the wave (SCHRADER and HELL 1996).

Fig. 6: Separation of excitation and fluorescence light in incident illumination. The CBS is placed at a 45° angle to the light path. This CBS reflects the bleu (wanted) excitation light onto the preparation and transmits the green fluorescence light towards the eyepieces. The blue (unwanted) excitation light is partly reflected backwards into the direction of the light source and only a small part is transmitted towards the eyepieces (modified from HELL 1997).

Materials and Methods

35 4.4 Radioimmunoassay of AVT

Arginine vasotocin radioimmunoassay was performed according to XU (1991) with some modifications. In short, aliquots of the chicken plasma samples (200µl) were extracted with cold acetone (-20°C) and centrifuged at 4.000 x g for 10 minutes at 4°C. The supernatant was decanted and mixed with 800 µl petroleum benzene and shaken 30 seconds and then left at room temperature for 30 minutes. The ether phase was discarded and the aqueous layer extracted once again with petroleum benzene. After discarding the ether phase, the aqueous phase was dried under vacuum in a SpeedVac (Savant, New York). The dried extract was stored at –20°C until assayed. The RIA was performed in duplicate using synthetic AVT (Sigma, Steinheim) as a standard. Just before conduction of the assay, the dried extracts were redissolved in 200µl of 0.1 M Tris-HCl, pH 7.4 and 200 µl of the antiserum were added. Standard curves were obtained by adding 200µl of dilutions of standard AVT and 200µl of the antiserum at working dilution giving a final dilution of 1:200.000.

The AVT antiserum was kindly supplied by Dr. Gray, Max-Planck-Institute for separation of bound and free tracer was done by the rapid addition of 800µl absolute ethanol followed by mixing and centrifugation at 3.500 rpm for 20 minutes at 4°C.

The supernatant were removed by aspiration and radioactivity in the pellets was read by using the gamma counter spectrometry (1277 Gamma Master, LKB Wallac). The results were analysed with RiaCalc Program (Pharmacia, LKB Wallac). The detection limit of the assay was 1.5 pg/ml. All samples were measured in the same assay. The intraassay variation coefficient was 7.2±0.2%.